Detection of hypermutable nucleic acid sequence in tissue

ABSTRACT

An assay for detection of a mammalian cell proliferative disorder associated with a hypermutable nucleic acid sequences is provided. The identification of particular hypermutable sequences such as microsatellite loci correlates with a particular cancer, thereby allowing detection of both primary tumors and metastatic sites within a patient.

RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 09/164,764, filedOct. 1, 1998, now issued as U.S. Pat. No. 6,479,234, which is acontinuation of U.S. Ser. No. 08/854,727, filed May 12, 1997, now issuedas U.S. Pat. No. 5,935,787, which is a continuation of U.S. Ser. No.08/299,477, filed Aug. 31, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to detection of a target nucleicacid sequence and specifically to detection of a cell proliferativedisorder associated with a hypermutable nucleic acid sequence in asample.

2. Description of Related Art

Mammalian genomes consist of unique DNA sequences interspersed withmoderately and highly repetitive DNA sequences. Gene mapping by meioticlinkage analysis has traditionally been carried out using variations inunique sequence DNA, such as restriction fragment length polymorphisms(Botstein, et al., Am. J. Hum. Genet., 32:314-331, 1980), as geneticmarkers. Recently, variations in the repetitive sequence elements suchas minisatellite or variable number tandem repeat (VNTR) sequences(Jeffreys, et al., Nature, 314:67-73, 1985; Nakamura, et al., Science,235:1616-1622, 1987), and microsatellite or variable simple sequencemotifs (VSSM) (Litt and Luty, Am. J. Hum. Genet., 44:397-401, 1989;Weber and May, Am. J. Hum. Genet., 44:388-396,-1989) have been found tobe useful for linkage studies. One advantage to the use of repetitivesequence variations rather than unique sequence variations is theapparently greater number of alleles present in normal populations whencompared to restriction fragment length polymorphisms (RFLPs). A secondadvantage is the ability to readily detect sequence length variationsusing the polymerase chain reaction to facilitate the rapid andinexpensive analysis of large numbers of DNA samples.

Microsatellite elements consist of simple mono-, di-, or tri-nucleotidesequences where alleles differ by one or more repeat units (Luty, etal., Am. J. Hum. Genet., 46:776-783, 1990; Tautz, et al., Nature,322:652-656, 1986; Weber and May, Am. J. Hum. Genet., 44:388-396, 1989).Minisatellites, or VNTR sequences, typically have a repeat unit of 20 toseveral hundred nucleotides and alleles differ by as little as onerepeat unit. Among simple sequences, the (TG)_(n) or (CA)_(n) repeatelements have recently proven extremely useful for meiotic mapping since(1) they are abundant in the genome, (2) display a large number ofdifferent alleles, and (3) can be rapidly assayed using the polymerasechain reaction (Litt and Luty, Am. J. Hum. Genet., 44:397-401, 1989;Weber and May, Am. J. Hum. Genet., 44:388-396, 1989).

A number of other short sequence motifs have been found in mammaliangenomes (Hellman, et al., Gene, 68:93-100, 1988; Knott, et al., Nuc.Acids Res., 14:9215-9216, 1986; Litt and Luty, Am. J. Hum. Genet.,44:397-401, 1989; Milstein, et al., Nuc. Acids Res., 12:6523-6535, 1984;Stoker, et al., Nuc. Acids Res., 13:4613-4621, 1985; Vassart, et al.,Science, 233:683-684, 1987; and Vergnaud, Nuc. Acids Res., 17:7623-7630,1989), and avian genomes (Gyllensten, et al., Nuc. Acids Res.,17:2203-2214, 1989; Longmire, et al., Genomics, 2:14-24, 1988) and arethought to accumulate by DNA slippage during replication (Tautz, et al.,Nature, 322:652-656, 1986) or unequal recombination events (Wolff, etal, Genomics, 5:382-384,1989). Many of these repeat elements display ahigh degree of genetic variation and, thus, are also useful for meioticand mitotic mapping.

The VNTR sequence isolated by Jeffreys, (supra) contains an invariantcore sequence GGGCAGGAXG (SEO ID NO: 41) which bears some similaritiesto the chi sequence of phage lambda (Wolff, et at., Genomics 5:382-384,1989) and is detected by a restriction fragment of bacteriophage M13(Vassart, et at., Science, 233:683-684, 1987). Similar repeat elementshave been detected by Nakamura, et at. (Science, 235:1616-1622, 1987)and contain a similar, but distinctive, common core unit GG—GGTGGGG (SEQID NO: 42). Elements of this type occur within several known genesequences including the β globin locus. Similar VNTR elements have beendescribed within the apolipoprotein B (Boerwinikle, et at., Proc. Natl.Acad. Sci. USA, 86:212-216, 1989; Knott, et al., Nuc. Acids Res.,14:9215-9216, 1986) and collagen type II genes (Stoker, et at., Nuc.Acids Res., 13:4613-4621, 1985) and contain a distinct AT-rich motif.Though a physiological function for repetitive elements of this type hasnot been defined, they have been suggested as potential hot spots forchromosome recombination (DeBustros, et at., Proc. Natl. Acad. Sci. USA85:5693-5697, 1988) or elements important for the control of geneexpression (Hellman, et al., Gene, 68:93-100, 1988; Milstein, et al.,Nuc. Acids Res., 12:6523-6535, 1984).

Microsatellites represent a very common and highly polymorphic class ofgenetic elements in the human genome. Microsatellite markers containingrepeat sequences have been used for primary gene mapping and linkageanalysis as described (Weber, et al., Am. J. Human Genet., 44:388,1989). PCR amplification of these repeats allows rapid assessment forloss of heterozygosity (LOH) and can greatly simplify procedures formapping tumor suppressor genes (Ruppert, et al., Cancer Res. 53:5093,1993; van der Riet, et al., Cancer Res., 54:1156, 1994). More recently,microsatellites have been used to identify specific mutations in certaininherited disorders including Huntington's disease (HD), fragile Xchromosome (FX), myotonic dystrophy (MD), spinocerebellar ataxia type I(SCA1), spino-bulbar muscular dystrophy (SBMA) and hereditarydentatorubralpallidoluysian atrophy (DRPLA) (The Huntington's DiseaseCollaborative Research Group., Cell, 72:971, 1993; E. J. Kremer, et al.,Science, 252:1711, 1991; G. Imbert, et al., Nature Genet., 4:72, 1993);H. T. Orr, et al., Nature Genet., 4:221, 1993); V. Biancalana, et al.,Hum. Mol. Genet., 1:255, 1992, M-Y., Chung, et al., Nature Genet.,5:254, 1993, R. Koide, et al., Nature Genet., 6:9, 1994).

Microsatellite instability has recently been described in human cancers.For example, microsatellite instability has been reported to be animportant feature of tumors from hereditary non-polyposis colorectalcarcinoma (HNPCC) patients (Peltomäki, et al., Science, 260:810, 1993;Aaltonen, et al., Science, 260:812, 1993; Thibodeau, et al., Science,260:816, 1993). Moreover, microsatellite instability, demonstrated byexpansion or deletion of repeat elements has been reported incolorectal, endometrial, breast, gastric, pancreatic, bladder neoplastictissues (J. I. Risinger, et al., Cancer Res., 53:5100, 1993; H-J. Han,et al., Cancer Res., 53:5087, 1993; P. Peltomäki, et al., Cancer Res.,53:5853, 1993; M. Gonzalez-Zulueta, et al., Cancer Res., 53:5620, 1993),and recently in SCLC. In HNPCC patients, this genetic instability is dueto inherited and somatic mutations of a critical mismatch repair gene(hMSH-2). Mutations of hMLH-1 and other critical mismatch repair genesmay also be responsible for the instability detected in HNPCC patients.

Cancer remains a major cause of mortality worldwide, and despiteadvancements in diagnosis and treatment, the overall survival rate hasnot improved significantly in the past twenty years. There remains anunfulfilled need for a more sensitive means of early diagnosis. Typicalassays to detect rare infiltrating tumor cells in clinical samplesutilize amplification methods that require additional cloning steps andsynthesis of a large number of oligomer specific probes to detect a widevariety of oncogenic mutations for each tumor type (Sidransky, et al.,Science, 252:706, 1991; Sidransky, et al., Science, 256:102, 1992). Thepresent invention provides a sensitive assay to detect a variety ofcancers using hypermutable microsatellite markers and an amplificationstrategy which eliminates the need for additional cloning steps.

SUMMARY OF THE INVENTION

The present invention provides a fast, reliable, sensitive screeningmethod for the detection of a cell proliferative disorder in variousclinical samples. The invention utilizes amplification of microsatellitenucleic acid (small repeat sequences) to detect a clonal population ofcells in a clinical sample.

The invention is based on the unexpected finding that microsatellitealterations are detectable as a clonal population of cells in the DNA ofcytologic clinical samples. These samples include urine, sputum, andhistopathologic margins obtained from cancer patients.

The invention provides a method for detecting a mammalian cellproliferative disorder (i.e., neoplasia) associated with a hypermutablemammalian target nucleic acid in a specimen, comprising isolating thenucleic acid present in the specimen and detecting the presence of thehypermutable target nucleic acid, typically following amplification ofthe nucleic acid.

In one embodiment, the amplification step in the method of the inventionis performed as a multiplex reaction. Therefore, instead of performingmultiple amplification reactions to identify each clonal alteration,primers for different markers are combined in one simple amplificationreaction, enhancing the identification of a large proportion of cellproliferative disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a denaturing acrylamide gel of normal (N) and tumor (T) DNAfrom lane 1, B17 (TCC) (marker FGA); lane 2, L21 (SCLC) (marker AR);lane 3, B30 (TCC) (marker UT762); and lane 4, L5 (SCLG) (marker D14S50).

FIG. 2 shows a denaturing acrylamide gel of Panel A) a urine (U) sampleof B27 (TCC) analyzed with marker FGA and compared with normal (N) andtumor (T) tissue DNA; Panel B) histological margin (M) of L31 (NSCLC)screened with AR and compared with tumor tissue; and Panel C) sputum (S)sample of L25 (SCLC) analyzed with CHRNB and compared with normal andtumor tissue DNA.

FIG. 3 shows DNA from patients' lymphocytes (N) amplified with markerFGA at varying dilutions with tumor DNA from 1 in 5 (20%) to 1 in 1000(0.1%). Novel bands are indicated by an arrow. Panel A, tumor DNA fromB17 (TCC); panel B, tumor DNA of B27 (TCC).

FIG. 4 shows a multiplex PCR assay utilizing DNA from B27 (TCC) and acorresponding urine sample amplified with three different primer sets(FGA, ACTBP2, AR) in the same PCR reaction (N: normal DNA; T: tumor DNA;U: urine DNA).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of detecting a cellproliferative disorder associated with a target nucleic acid having ahypermutable nucleotide sequence. Preferably, the hypermutablenucleotide sequence of the invention is a microsatellite DNA sequence.Microsatellite alterations in various cancer lesions, for example, canbe detected by using known microsatellite repeat markers. A combinationof repeat markers can be utilized in the method of the invention toidentify a large percentage of cell proliferative disorders orneoplasias even if the neoplastic cells comprise only a small fractionof the clinical sample.

As used herein, the term “hypermutable” refers to a nucleic acidsequence that is susceptible to instability, thus resulting in nucleicacid alterations. Such alterations include the deletion and addition ofnucleotides. The hypermutable sequences of the invention are preferablymicrosatellite DNA sequences which, by definition, are small tandemrepeat DNA sequences.

The hypermutable nucleic acid may be a neoplastic nucleic acid sequence.The term “neoplastic” nucleic acid refers to a nucleic acid sequencewhich directly or indirectly is associated with or causes a neoplasm.The method of the invention is applicable to detection of hypermutablenucleotide sequences associated with benign as well as malignant tumors.The method can be used to detect any hypermutable nucleotide sequence,regardless of origin, as long as the sequence is detectably present in aspecimen. The specimen can be blood, urine, sputum, bile, stool,cervical smears, saliva, tears, cerebral spinal fluid, regional lymphnode and histopathologic margins, and any bodily fluid that drains abody cavity or organ. For example, neoplasia of regional lymph nodesassociated with a primary mammary tumor can be detected utilizing themethod of the invention. The term “regional lymph node” refers tolymphoid tissue forming lymphoid organs or nodes which are in closeproximity to the primary tumor. For example, regional lymph nodes in thecase of head and neck carcinomas include cervical lymph nodes,prelaryngeal lymph nodes, pulmonary juxtaesophageal lymph nodes andsubmandibular lymph nodes. Regional lymph nodes for mammary tissuecarcinomas include the axillary and intercostal nodes. The term“external to a primary neoplasm” means that the specimen is taken from asite other than directly from the primary neoplasm itself. Such specimenmay be useful in evaluating whether metastasis of the primary neoplasmhas occurred.

The method can also be used to detect a hypermutable nucleic acidsequence associated with a primary tumor by assaying the surroundingtumor margin. As used herein the term “tumor margin” refers to thetissue surrounding a discernible tumor. In the case of surgical removalof a solid tumor, the tumor margin is the tissue cut away with thediscernible tumor that usually appears to be normal to the naked eye.More particularly, as used herein, “margin” refers to the edge, border,or boundary of a tumor. The margin generally extends from about 0.2 cmto about 3 cm from the primary tumor but can be greater depending uponthe size of the primary solid tumor.

In its broadest sense, the present invention allows the detection of anyhypermutable target nucleic acid sequence of diagnostic or therapeuticrelevance, where the target nucleic acid sequence is present in a tissuesample. The target nucleotide sequence may be, for example, arestriction fragment length polymorphism (RFLP), nucleotide deletion,nucleotide addition, or any other mammalian nucleic acid sequence ofinterest in such tissue specimens. Preferably, the microsatellite,hypermutable nucleic acid of the invention contains nucleic aciddeletions or additions.

The hypermutable microsatellites most preferred in the method of theinvention comprises the sequence (X)_(n), wherein X is the number ofnucleotides in the repeat sequence and is greater than or equal to 1,preferably greater than or equal to 2, and most preferably greater thanor equal to 3 and wherein n is the number of repeats and is greater thanor equal to 2, and preferably from 4 to 6. Preferably, when X is 2, thenucleotide sequence is TC. Preferably, when X is 3, the nucleotidesequence is selected from AGC, TCC, CAG, CM, and CTG. Preferably when Xis 4, the nucleotide sequence is selected from AAAG, AGAT and TCTT.

The hypermutable nucleic acid sequence is preferably associated with aknown locus. For example, hypermutable microsatellite alterations may bedetected using a marker selected from ARA (chromosome X), D14S50(chromosome 14), AR (chromosome X), MD (chromosome 19), SAT (chromosome6), DRPLA (chromosome 12), ACTBP2 (chromosome 6), FGA (chromosome 4),D4S243 (chromosome 4), and UT762 (chromosome 21). Tandem repeatsequences have been identified as associated with Huntington's disease(HD), fragile X syndrome (FX), myotonic dystrophy (MD), spinocerebellarataxia type I (SCAl), spino-bulbar muscular dystrophy, and hereditarydentatorubralpallidoluysian atrophy (DRPLA). When the nucleotidesequence of X is larger, it is more likely that the microsatellite locuswill have alterations, e.g., a trinucleotide repeat is more likely tohave deletions or additions than a dinucleotide repeat. Thus, in thepresent invention, it has been found that 8% of 25 trinucleotide ortetranucleotide markers displayed microsatellite alternations per 100tumor specimens examined, whereas only 0.7% of 83 dinucleotidemicrosatellite markers were found to contain alterations. In addition,it has been found that a regular repeat, such as AAT AAT AAT is morelikely to be hypermutable than a sequence which contains interruptionsin the repeat sequence, e.g., AAT GAC AAT AAT (SEO ID NO: 43).Consequently, those of ordinary skill in the art can readily identifyother hypermutable target nucleic acid sequences by considering the sizeof the candidate sequence and whether the sequence is uninterruptedwithout resorting to undue experimentation. Other microsatellite markerswill be known by the criteria described herein and are accessible tothose of skill in the art. Smaller microsatellite markers includingdinucleotide and mononucleotide repeats may be hypermutable and usefulfor this analysis.

The present invention identifies hypermutable target sequences,preferably microsatellite loci, that are unique to a particular cellularproliferative disorder, primary tumor, or metastatic sites derived fromthe primary tumor. In the tumor cell, the hypermutable nucleotidesequence is evidenced by nucleic acid deletions or expansion of repeatsequences as compared to a normal cell; therefore, it is possible todesign appropriate diagnostic techniques directed to the specificsequence and to design therapeutic strategies once the sequence isidentified.

The term “cell-proliferative disorder” denotes benign as well asmalignant cell populations which morphologically often appear to differfrom the surrounding tissue. For example, the method of the invention isuseful in detecting malignancies of the various organ systems, such as,for example, lung, breast, lymphoid, gastrointestinal, andgenito-urinary tract as well as epithelial carcinomas which includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer, non-small cell carcinoma of the lung, cancer of the smallintestine, cancer of the head and neck, stomach cancer, bladder cancer,kidney cancer, cervical cancer, cancer of the esophagus and any otherorgan type that has a draining fluid or tissue accessible to analysis.The method of the invention is also useful in detecting non-malignantcell-proliferative diseases such as colon adenomas, hyperplasia,dysplasia and other “pre-malignant” lesions. Essentially, any disorderwhich is etiologically linked to a hypermutable microsatellite locuswould be considered susceptible to detection.

When it is desired to amplify the target nucleotide sequence beforedetection, such as a hypermutable nucleotide sequence, this can beaccomplished using oligonucleotides that are primers for amplification.The oligonucleotide primers are designed based upon identification ofthe nucleic acid sequence of the flanking regions contiguous with thehypermutable nucleotide sequence. For example, in the case ofhypermutable microsatellite nucleic acid sequences, oligonucleotideprimers comprise sequences which are capable of hybridizing withnucleotide sequences flanking the loci of mutations, such as thefollowing nucleotide sequences:

a. 5′-CTT GTG TCC CGG CGT CTG-3′; (SEQ ID NO:1) b. 5′-C AGC CCA GCA GGACCA GTA-3′; (SEQ ID NO:2) c. 5′-TGG TAA CAG TGG AAT ACT GAC-3′; (SEQ IDNO:3) d. 5′-ACT GAT GCA AAA ATC CTC AAC-3′; (SEQ ID NO:4) e. 5′-GA TGGGCA AAC TGC AGG CCT GGG AAG-3′; (SEQ ID NO:5) f. 5′-GCT ACA AGG ACC CTTCGA GCC CCG TTC-3′; (SEQ ID NO:6) g. 5′-GAT GGT GAT GTG TTG AGA CTGGTG-3′; (SEQ ID NO:7) h. 5′-GAG CAT TTC CCC ACC CAC TGG AGG-3′; (SEQ IDNO:8) i. 5′-GTT CTG GAT CAC TTC GCG GA-3′; (SEQ ID NO:9) j. 5′-TGA GGATGG TTC TCC CCA AG-3′; (SEQ ID NO:10) k. 5′-AGT GGT GAA TTA GGG GTGTT-3′; (SEQ ID NO:11) l. 5′-CTG CCA TCT TGT GGA ATC AT-3′; (SEQ IDNO:12) m. 5′-CTG TGA GTT CAA AAC CTA TGG-3′; (SEQ ID NO:13) n. 5′-GTGTCA GAG GAT CTG AGA AG-3′; (SEQ ID NO:14) o. 5′-GCA CGC TCT GGA ACA GATTCT GGA-3′; (SEQ ID NO:15) p. 5′-ATG AGG AAC AGC AAC CTT CAC AGC-3′;(SEQ ID NO:16) q. 5′-TCA CTC TTG TCG CCC AGA TT-3′; (SEQ ID NO:17) r.5′-TAT AGC GGT AGG GGA GAT GT-3′; (SEQ ID NO:18) s. 5′-TGC AAG GAG AAAGAG AGA CTG A-3′; (SEQ ID NO:19) t. 5′-AAC AGG ACC ACA GGC TCC TA-3′;and (SEQ ID NO:20) u. sequences complementary to sequences a. through t.

Primers that hybridize to these flanking sequences are, for example, thefollowing:

a. 5′-CAG ACG CCG GGA CAC AAG-3′; (SEQ ID NO:21) b. 5′-TAC TGG TCC TGCTGG GCT G-3′; (SEQ ID NO:22) c. 5′-GTC AGT ATT ACC CTG TTA CCA-3′; (SEQID NO:23) d. 5′-GTT GAG GAT TTT TGC ATC AGT-3′; (SEQ ID NO:24) e. 5′-CTTCCC AGG CCT GCA GTT TGC CCA TC-3′; (SEQ ID NO:25) f. 5′-GAA CGG GGC TCGAAG GGT CCT TGT AGC-3′; (SEQ ID NO:26) g. 5′-CAC CAG TCT CAA CAC ATC ACCATC-3′; (SEQ ID NO:27) h. 5′-CCT CCA GTG GGT GGG GAA ATG CTC-3′; (SEQ IDNO:28) i. 5′-TCC GCG AAG TGA TCC AGA AC-3′; (SEQ ID NO:29) j. 5′-CTT GGGGAG AAC CAT CCT CA-3′; (SEQ ID NO:30) k. 5′-AAC ACC CCT AAT TCA CCACT-3′; (SEQ ID NO:31) l. 5′-ATG ATT CCA CAA GAT GGC AG-3′; (SEQ IDNO:32) m. 5′-CCA TAG GTT TTG AAC TCA CAG-3′; (SEQ ID NO:33) n. 5′-CTTCTC AGA TCC TCT GAC AC-3′; (SEQ ID NO:34) o. 5′-TCC AGA ATC TGT TCC AGAGCG TGC-3′; (SEQ ID NO:35) p. 5′-GCT GTG AAG GTT GCT GTT CCT CAT-3′;(SEQ ID NO:36) q. 5′-AAT CTG GGC GAC AAG AGT GA-3′; (SEQ ID NO:37) r.5′-ACA TCT CCC CTA CCG CTA TA-3′; (SEQ ID NO:38) s. 5′-TCA GTC TCT CTTTCT CCT TGC A-3′; (SEQ ID NO:39) t. 5′-TAG GAG CCT GTG GTC CTG TT-3′;and (SEQ ID NO:40) u. sequences complementary to sequences a. through t.

One skilled in the art will be able to generate primers suitable foramplifying target sequences of additional nucleic acids, such as thoseflanking loci of known microsatellite sequences, using routine skillsknown in the art and the teachings of this invention.

In general, the primers used according to the method of the inventionembrace oligonucleotides of sufficient length and appropriate sequencewhich provide specific initiation of polymerization of a significantnumber of nucleic acid molecules containing the target nucleic acidunder the conditions of stringency for the reaction utilizing theprimers. In this manner, it is possible to selectively amplify thespecific target nucleic acid sequence containing the nucleic acid ofinterest. Specifically, the term “primer” as used herein refers to asequence comprising two or more deoxyribonucleotides or ribonucleotides,preferably at least eight, which sequence is capable of initiatingsynthesis of a primer extension product that is substantiallycomplementary to a target nucleic acid strand. The oligonucleotideprimer typically contains 15-22 or more nucleotides, although it maycontain fewer nucleotides as long as the primer is of sufficientspecificity to allow essentially only the amplification of thespecifically desired target nucleotide sequence (i.e., the primer issubstantially complementary).

Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization, such as DNApolymerase, and a suitable temperature and pH. The primer is preferablysingle stranded for maximum efficiency in amplification, but may bedouble stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent for polymerization. The exact length ofprimer will depend on many factors, including temperature, buffer, andnucleotide composition.

Primers used according to the method of the invention are designed to be“substantially” complementary to each strand of mutant nucleotidesequence to be amplified. Substantially complementary means that theprimers must be sufficiently complementary to hybridize with theirrespective strands under conditions which allow the agent forpolymerization to function. In other words, the primers should havesufficient complementarily with the flanking sequences to hybridize withand permit amplification of the nucleotide sequence. Preferably, the 3′terminus of the primer that is extended has perfectly base pairedcomplementarity with the complementary flanking strand.

Oligonucleotide primers used according to the invention are employed inany amplification process that produces increased quantities of targetnucleic acid. Typically, one primer is complementary to the negative (−)strand of the mutant nucleotide sequence and the other is complementaryto the positive (+) strand. Annealing the primers to denatured nucleicacid followed by extension with an enzyme, such as the large fragment ofDNA Polymerase I (Klenow) or Taq DNA polymerase and nucleotides orligases, results in newly synthesized (+) and (−) strands containing thetarget nucleic acid. Because these newly synthesized nucleic acids arealso templates, repeated cycles of denaturing, primer annealing, andextension results in exponential production of the region (i.e., thetarget hypermutable nucleotide sequence) defined by the primer. Theproduct of the amplification reaction is a discrete nucleic acid duplexwith termini corresponding to the ends of the specific primers employed.Those of skill in the art will know of other amplification methodologieswhich can also be utilized to increase the copy number of target nucleicacid.

The oligonucleotide primers for use in the invention may be preparedusing any suitable method, such as conventional phosphotriester andphosphodiester methods or automated embodiments thereof. In one suchautomated embodiment, diethylphosphoramidites are used as startingmaterials and may be synthesized as described by Beaucage, et al.(Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. One method of amplification which can be used accordingto this invention is the polymerase chain reaction (PCR) described inU.S. Pat. Nos. 4,683,202 and 4,683,195.

The nucleic acid from any specimen, in purified or nonpurified form, canbe utilized as the starting nucleic acid or acids, provided it contains,or is suspected of containing, the specific nucleic acid sequencecontaining the target nucleic acid. Thus, the process may employ, forexample, DNA or RNA, including messenger RNA (mRNA), wherein DNA or RNAmay be single stranded or double stranded. In the event that RNA is tobe used as a template, enzymes, and/or conditions optimal for reversetranscribing the template to DNA would be utilized. In addition, aDNA-RNA hybrid which contains one strand of each may be utilized. Amixture of nucleic acids may also be employed, or the nucleic acidsproduced in a previous amplification reaction herein, using the same ordifferent primers may be so utilized. The mutant nucleotide sequence tobe amplified may be a fraction of a larger molecule or can be presentinitially as a discrete molecule, such that the specific sequenceconstitutes the entire nucleic acid. It is not necessary that thesequence to be amplified be present initially in a pure form; it may bea minor fraction of a complex mixture, such as contained in whole humanDNA.

Where the target neoplastic nucleotide sequence of the sample containstwo strands, it is necessary to separate the strands of the nucleic acidbefore it can be used as the template. Strand separation can be effectedeither as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing various suitable denaturing conditions, including physical,chemical, or enzymatic means; the word “denaturing” includes all suchmeans. One physical method of separating nucleic acid strands involvesheating the nucleic acid until it is denatured. Typical heatdenaturation may involve temperatures ranging from about 80° to 105° C.for times ranging from about 1 to 10 minutes. Strand separation may alsobe induced by an enzyme from the class of enzymes known as helicases orby the enzyme RecA, which has helicase activity, and in the presence ofriboATP which is known to denature DNA. The reaction conditions suitablefor strand separation of nucleic acids with helicases are described byKuhn Hoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) andtechniques for using RecA are reviewed in C. Radding (Ann. Rev.Genetics, 16:405-437, 1982).

If the nucleic acid containing the target nucleic acid to be amplifiedis single stranded, its complement is synthesized by adding one or twooligonucleotide primers. If a single primer is utilized, a primerextension product is synthesized in the presence of primer, an agent forpolymerization, and the four nucleoside triphosphates described below.The product will be complementary to the single-stranded nucleic acidand will hybridize with a single-stranded nucleic acid to form a duplexof unequal length strands that may then be separated into single strandsto produce two single separated complementary strands. Alternatively,two primers may be added to the single-stranded nucleic acid and thereaction carried out as described.

When complementary strands of nucleic acid or acids are separated,regardless of whether the nucleic acid was originally double or singlestranded, the separated strands are ready to be used as a template forthe synthesis of additional nucleic acid strands. This synthesis isperformed under conditions allowing hybridization of primers totemplates. Generally synthesis occurs in a buffered aqueous solution,preferably at a pH of 7-9, most preferably about 8. Preferably, a molarexcess (for genomic nucleic acid, usually about 10⁸:1 primer:template)of the two oligonucleotide primers is added to the buffer containing theseparated template strands. It is understood, however, that the amountof complementary strand may not be known if the process of the inventionis used for diagnostic applications, so that the amount of primerrelative to the amount of complementary strand cannot be determined withcertainty. As a practical matter, however, the amount of primer addedwill generally be in molar excess over the amount of complementarystrand (template) when the sequence to be amplified is contained in amixture of complicated long-chain nucleic acid strands. A large molarexcess is preferred to improve the efficiency of the process.

In some amplification embodiments, the substrates, for example, thedeoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP, are addedto the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period, the solution is allowed to cool toroom temperature, which is preferable for the primer hybridization. Tothe cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein “agent for polymerization”),and the reaction is allowed to occur under conditions known in the art.The agent for polymerization may also be added together with the otherreagents if it is heat stable. This synthesis (or amplification)reaction may occur at room temperature up to a temperature above whichthe agent for polymerization no longer functions. Thus, for example, ifDNA polymerase is used as the agent, the temperature is generally nogreater than about 40° C. The agent for polymerization may be anycompound or system which will function to accomplish the synthesis ofprimer extension products, including enzymes. Suitable enzymes for thispurpose include, for example, E. coli DNA polymerase I, Taq polymerase,Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, otheravailable DNA polymerases, polymerase muteins, reverse transcriptase,ligase, and other enzymes, including heat-stable enzymes (i.e., thoseenzymes which perform primer extension after being subjected totemperatures sufficiently elevated to cause denaturation). Suitableenzymes will facilitate combination of the nucleotides in the propermanner to form the primer extension products which are complementary toeach mutant nucleotide strand. Generally, the synthesis will beinitiated at the 3′ end of each primer and proceed in the 5′ directionalong the template strand, until synthesis terminates, producingmolecules of different lengths. There may be agents for polymerization,however, which initiate synthesis at the 5′ end and proceed in the otherdirection, using the same process as described above. In any event, themethod of the invention is not to be limited to the embodiments ofamplification which are described herein.

The newly synthesized mutant nucleotide strand and its complementarynucleic acid strand will form a double-stranded molecule underhybridizing conditions described above and this hybrid is used insubsequent steps of the process. In the next step, the newly synthesizeddouble-stranded molecule is subjected to denaturing conditions using anyof the procedures described above to provide single-stranded molecules.

The above process is repeated on the single-stranded molecules.Additional agent for polymerization, nucleotides, and primers may beadded, if necessary, for the reaction to proceed under the conditionsprescribed above. Again, the synthesis will be initiated at one end ofeach of the oligonucleotide primers and will proceed along the singlestrands of the template to produce additional nucleic acid. After thisstep, half of the extension product will consist of the specific nucleicacid sequence bounded by the two primers.

The steps of denaturing and extension product synthesis can be repeatedas often as needed to amplify the target hypermutable nucleotidesequence to the extent necessary for detection. The amount of thehypermutable nucleotide sequence produced will accumulate in anexponential fashion.

In one embodiment of the invention, a combination of hypermutablemicrosatellite markers are amplified in a single amplification reaction.The markers are “multiplexed” in a single amplification reaction, forexample, by combining primers for more than one locus. For example, DNAfrom a urine sample is amplified with three different randomly labelledprimer sets such as FGA, ACTBP2 and AR, in the same amplificationreaction. The products are ultimately separated on a denaturingacrylamide gel and then exposed to film for visualization and analysis.

The amplified product may be detected by Southern blot analysis, withoutusing radioactive probes. In such a process, for example, a small sampleof DNA containing a very low level of microsatellite hypermutablenucleotide sequence is amplified, and analyzed via a Southern blottingtechnique. The use of non-radioactive probes or labels is facilitated bythe high level of the amplified signal. In a preferred embodiment of theinvention, one nucleoside triphosphate is radioactively labeled, therebyallowing direct visualization of the amplification product byautoradiography. In another embodiment, amplification primers arefluorescent labelled and run through an electrophoresis system.Visualization of amplified products is by laser detection followed bycomputer assisted graphic display.

Nucleic acids having a hypermutable microsatellite sequence detected inthe method of the invention can be further evaluated, detected, cloned,sequenced, and the like, either in solution or after binding to a solidsupport, by any method usually applied to the detection of a specificDNA sequence such as PCR, oligomer restriction (Saiki, et al.,Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide(ASO) probe analysis (Conner, et al., Proc. Natl. Acad. Sci. USA,80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, etal., Science, 241:1077, 1988), and the like. Molecular techniques forDNA analysis have been reviewed (Landegren, et al., Science,242:229-237, 1988).

In another embodiment of the invention, purified nucleic acid fragmentsthat contain oligonucleotide sequences of 10-50 bases frommicrosatellite loci, are radioactively labelled. The labelledpreparations are used to probe nucleic acid by the Southernhybridization technique. Nucleotide fragments from a specimen, before orafter amplification, are separated into fragments of different molecularmasses by gel electrophoresis and transferred to filters which bindnucleic acid. After exposure to the labelled probe, which will hybridizeto nucleotide fragments containing target nucleic acid sequences,binding of the radioactive probe to target nucleic acid fragments isidentified by autoradiography (see Genetic Engineering, 1, ed. RobertWilliamson, Academic Press, (1981), 72-81).

Probes for microsatellite loci of the present invention can be used forexamining the distribution of the specific fragments detected, as wellas the quantitative (relative) degree of binding of the probe fordetermining the occurrence of specific strongly binding (hybridizing)sequences, thus indicating the presence of extensive alterations in aparticular locus.

For the most part, the probe (or amplification primer) will be labelledwith an atom or inorganic radical, most commonly using radionuclides,but also perhaps heavy metals. Conveniently, a radioactive label may beemployed. Radioactive labels include ³²P, ¹²⁵I, ³H, ¹⁴C, ³⁵S, or thelike. Any radioactive label may be employed which provides for anadequate signal and has sufficient half-life. Other labels includeligands, which can serve as a specific binding pair member for alabelled ligand, and the like. A wide variety of labels have beenemployed in immunoassays which can readily be employed in the presentassay. The choice of the label will be governed by the effect of thelabel on the rate of hybridization and binding of the probe to mutantnucleotide sequence. It will be necessary that the label providesufficient sensitivity to detect the amount of mutant nucleotidesequence available for hybridization. Other considerations will be easeof synthesis of the probe, readily available instrumentation, ability toautomate, convenience, and the like.

The manner in which the label is bound to the probe will vary dependingupon the nature of the label. For a radioactive label, a wide variety oftechniques can be employed. Commonly employed is nick translation withan α-³²P-dNTP or terminal phosphate hydrolysis with alkaline phosphatasefollowed by labeling with radioactive ³²P employing γ-³²P-ATP and T4polynucleotide kinase. Alternatively, nucleotides can be synthesizedwhere one or more of the elements present are replaced with aradioactive isotope, e.g., hydrogen with tritium. If desired,complementary labelled strands can be used as probes to enhance theconcentration of hybridized label.

Where other radionuclide labels are involved, various linking groups canbe employed. A terminal hydroxyl can be esterified, with inorganicacids, e.g., ³²P phosphate, or ¹⁴C organic acids, or else esterified toprovide linking groups to the label. Alternatively, intermediate basesmay be substituted with activatable linking groups which can then belinked to a label.

Enzymes of interest as reporter groups will primarily be alkalinephosphatase, hydrolases, particularly esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, and so forth. Chemiluminescers include, for example,luciferin, and 2,3-dihydrophthalazinediones (e.g., luminol).

An oligomer probe can be employed for hybridizing to a nucleotidesequence affixed to a water insoluble porous support. Depending upon thesource of the nucleic acid, the manner in which the nucleic acid isaffixed to the support may vary. Those of ordinary skill in the artknow, or can easily ascertain, different supports which can be used inthe method of the invention.

The nucleic acid from a specimen is spotted or spread onto a filter toprovide a plurality of individual portions. The filter is an inertporous solid support, e.g., nitrocellulose or nylon membranes. Anymammalian cells present in the specimen are treated to liberate theirnucleic acid. The lysing and denaturation of nucleic acid, as well asthe subsequent washings, can be achieved with an appropriate solutionfor a sufficient time to lyse the cells and denature the nucleic acid.Other denaturation agents include elevated temperatures, organicreagents (e.g., alcohols, amides, amines, ureas, phenols and sulfoxides)or certain inorganic ions (e.g., thiocyanate and perchlorate).Alternatively, nucleic acid can be isolated from blood using standardprocedures with the DNA subsequently applied to the membrane.

After denaturation, the filter is washed in an aqueous bufferedsolution, such as Tris, generally at a pH of about 6 to 8, usually 7.One or more washings may be involved, conveniently using the sameprocedure as employed for the lysing and denaturation. After the lysing,denaturing, and washes have been accomplished, the nucleic acid spottedfilter is dried at an elevated temperature, generally from about 50° C.to 70° C. or UV-crosslinked (for nylon membranes). Under this procedure,the nucleic acid is fixed in position and can be assayed with the probewhen convenient.

Pre-hybridization may be accomplished by incubating the filter at amildly elevated temperature for a sufficient time with the hybridizationsolution without the probe to thoroughly wet the filter. Varioushybridization solutions may be employed, comprising from about 20% to60% volume, preferably 30%, of an inert polar organic solvent or aqueoushybridization solutions.

The particular hybridization technique is not essential to theinvention. Other hybridization techniques are well known or easilyascertained by one of ordinary skill in the art. As improvements aremade in hybridization techniques they can readily be applied in themethod of the invention.

The amount of labelled probe which is present in the hybridizationsolution will vary widely, depending upon the nature of the label, theamount of the labelled probe which can reasonably bind to the filter,and the stringency of the hybridization. Generally, substantial excessover stoichiometric concentrations of the probe will be employed toenhance the rate of binding of the probe to the fixed target nucleicacid.

Various degrees of stringency of hybridization may be employed. The morestringent the conditions, the greater the complementarity that isrequired for hybridization between the probe and the single strandedtarget nucleic acid sequence for duplex formation. Stringency can becontrolled by temperature, probe concentration, probe length, ionicstrength, time, and the like. Conveniently, the stringency ofhybridization is varied by changing the polarity of the reactantsolution by manipulating the concentration of formamide in the range of20% to 50%. Temperatures employed will normally be in the range of about20° C. to 80° C., usually 30° C. to 75° C. (see, generally, CurrentProtocols in Molecular Biology, Ausubel, ed., Wiley & Sons, 1989).Alternatively, stringency can be controlled when the non-annealed probeis washed off.

After the filter has been contacted with a hybridization solution at amoderate temperature for a period of time sufficient to allowhybridization to occur, the filter is then introduced into a secondsolution having sodium chloride, sodium citrate and sodiumdodecylsulfate. The time for which the filter is maintained in thesecond solution may vary from five minutes to three hours or more. Thesecond solution and the temperature (generally 5° C. below the meltingtemperature) determines the stringency, dissolving duplexes, and shortcomplementary sequences. For short oligonucloetide probes, the meltingtemperature can be standarized according to probe length, rather thansequence, by including tetramethyl ammonium chloride in the washsolution (DiLella and Woo, Meth. Enzymol., 152:447, 1987). The filtermay now be assayed for the presence of duplexes in accordance with thenature of the label. Where the label is radioactive, the filter is driedand exposed to X-ray film.

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method.

For example, one of the container means may comprise amplificationprimers for a microsatellite locus or a hybridization probe, all ofwhich can be detectably labelled. If present, a second container maycomprise a lysis buffer. The kit may also have containers containingnucleotide(s) for amplification of the target nucleic acid sequencewhich may or may not be labeled, and/or a container comprising areporter-means, such as a biotin-binding protein, such as avidin orstreptavidin, bound to a reporter molecule, such as an enzymatic,florescent, or radionuclide label.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLES

During primary mapping studies using 43 dinucleotide markers in over 300tumors including SCC, non-small cell lung carcinoma (NSCLC),transitional cell carcinoma of the bladder (TCC), and squamous and basalcell skin carcinoma, single somatic alterations were observed inapproximately 0.7% of tumor DNA This low rate of “background”alterations in dinucleotide markers is consistent with other studiesdemonstrating few changes in non-HNPCC-associated tumors (S. N.Thibodeau, et al., Science, 260:816, 1993; J. I. Risinger, et al.,Cancer Res., 53:5100, 1993; H-J. Han, et al., Cancer Res., 53:5087,1993); P. Peltomaki, et al., Cancer Res., 53: 5853, 1993); M.Gonzalez-Zulueta, et al., Cancer Res., 53:5620, 1993; R. Wooster, etal., Nature Gent., 6:152, 1994) and germline DNA (J. L Weber, et al.,Am. J. Human Genet, 44:388, 1989), J. Weissenback, et al., Nature,359:794, 1992; (A. J. Jeffreys, et al., Nature Genet., 6:136, 1994). 35SCC, 20 NSCLC, 10 SCLC, and 32 TCC normal and cancer DNA pairs weretested using tri-and tetranucleotide repeat markers (TABLE 1). Of these,approximately 8% and 20% of tumor DNA had somatic alterations using tri-and tetranucleotide repeat-markers, respectively. Of the nine markers,four (MD, DRPLA, AR (SBMA), and SAT (SCA1)) were associated withneurological disease and have revealed germline expansion of theirrepeat sequences in affected patients. Because of these germlinealterations, it was thought that these markers might be more susceptibleto expansion or deletion in tumor DNA as compared with dinucleotiderepeat sequences. The other markers were chosen among commerciallyavailable tri- or tetranucleotide repeat markers except for UT762 (onhuman chromosome 21), which was previously reported to have an excess ofgermline alterations on human chromosome 21 (C. C. Talbot, Jr., et al.,43rd American Society of Human Genetics Meeting, Abstract, 1993).

Example 1 Primers Utilized for Amplification of Specimen DNA

All tumors were fresh-frozen except for SCLC which wasparaffin-embedded. Non-neoplastic tissue was microdissected away to beused as normal DNA. Alternatively, fresh blood was obtained andlymphocytes separated. Tumor and normal tissue was digested with 1%SDS-proteinase K followed by ethanol precipitation to extract DNA. 50 ngof DNA was subject to PCR amplification; the products were run ondenaturing acrylamide gels as described previously (P. van der Riet, etal., Cancer Res., 54-1156, 1994), H. Nawroz, P., Cancer Res., 54:1152,1994; P. Cairns, et al., Cancer Res., 54:1422, 1994).

Primers used to amplify each locus were obtained from Research Genetics,Inc., with the exception of the following loci:

a. 5′-CAG ACG CCG GGA CAC AAG-3′; (SEQ ID NO:21) b. 5′-TAC TGG TCC TGCTGG GCT G-3′; (SEQ ID NO:22) c. 5′-GTC AGT ATT ACC CTG TTA CCA-3′; (SEQID NO:23) d. 5′-GTT GAG GAT TTT TGC ATC AGT-3′; (SEQ ID NO:24) e. 5′-CTTCCC AGG CCT GCA GTT TGC CCA TC-3′; (SEQ ID NO:25) f. 5′-GAA CGG GGC TCGAAG GGT CCT TGT AGC-3′; (SEQ ID NO:26) g. 5′-CAC CAG TCT CAA CAC ATC ACCATC-3′; (SEQ ID NO:27) h. 5′-CCT CCA GTG GGT GGG GAA ATG CTC-3′; (SEQ IDNO:28) i. 5′-TCC GCG AAG TGA TCC AGA AC-3′; (SEQ ID NO:29) j. 5′-CTT GGGGAG AAC CAT CCT CA-3′; (SEQ ID NO:30) k. 5′-AAC ACC CCT AAT TCA CCACT-3′; (SEQ ID NO:31) l. 5′-ATG ATT CCA CAA GAT GGC AG-3′; (SEQ IDNO:32) m. 5′-CCA TAG GTT TTG AAC TCA CAG-3′; (SEQ ID NO:33) n. 5′-CTTCTC AGA TCC TCT GAC AC-3′; (SEQ ID NO:34) o. 5′-TCC AGA ATC TGT TCC AGAGCG TGC-3′; (SEQ ID NO:35) p. 5′-GCT GTG AAG GTT GCT GTT CCT CAT-3′;(SEQ ID NO:36) q. 5′-AAT CTG GGC GAC AAG AGT GA-3′; (SEQ ID NO:37) r.5′-ACA TCT CCC CTA CCG CTA TA-3′; (SEQ ID NO:38) s. 5′-TCA GTC TCT CTTTCT CCT TGC A-3′; (SEQ ID NO:39) t. 5′-TAG GAG CCT GTG GTC CTG TT-3′;and (SEQ ID NO:40) u. sequences complementary to sequences a. through t.

P. Modrich, Annu. Rev. Genet, 25:229, (1991). Cytological samples werespun at 3000×g for 5′, and washed with PBS twice. Cell pellets weredigested with 1% SDS-proteinase K, and DNA extracted as describedpreviously (D. Sidransky, et al., Science, 252:706, 1991), Science,256:102, 1992; Surgical margin DNA was obtained from slides which werehistopathologically negative. Tissue was scraped and placed in xylene toremove excess paraffin. After centrifugation with one quarter volume 70%ethanol, pellets were digested and DNA extracted as described previously(D. Sidransky, supra, R. H. Hruban, supra, L. Mao, supra).

Example 2 Detection of Alterations in Microsatellite LOCI

Each microsatellite locus was amplified in paired normal/tumor DNA byPCR and labelled products were then run on denaturing acrylamide gelsand exposed to film. 29% of head and neck cancers, 5% of NSCLC, 50% ofSCLC, and 28% of bladder tumors exhibited microsatellite alterations inat least one susceptible marker (TABLE 1). These genetic alterationswere identified as a novel band (or bands) in the tumor DNA lane andwere not present in the paired normal DNA lane (FIG. 1). FIG. 1 showsmicrosatellite alterations in tumor DNA. Normal and tumor DNA wereamplified by PCR and run on denaturing acrylamide gels as described (P.van der Riet, supra). Novel bands representing deletion or expansion oftandem repeat sequences were seen in all four tumor lanes as indicatedby the arrows. Lane 1) B17 (TCC) with marker FGA on chromosome 4; Lane2) L21 (SCLC) with marker AR on chromosome X; Lane 3) B30 (TCC) withmarker UT762 on chromosome 21; and Lane 4) L5 (SCLC) with marker D14S50on chromosome 14. (N: normal DNA. T: tumor DNA).

For each case, the amplification was repeated and the alterationsreproduced. The frequency of alterations was significantly higher inthese tri- or tetranucleotide repeat markers and also appeared to betumor-type specific (TABLE 1). In TCC, for example, the AR repeat isaltered in 3% of tumors whereas 18% of SCC's displayed alterations atthis locus. A significant difference in the frequency of alterationsbetween disease and non-disease related tri- and tetranucleotide repeatsequences was not observed suggesting that there might exist a moregeneralized cellular mechanism for these changes, rather than inherentsequence differences in the repeat regions. SCLC displayed generalizedmicrosatellite instability similar to that seen in HNPCC-associatedtumors and altered a high percentage of all markers includingdinucleotides (A. Merlo, et al., supra).

Although widespread microsatellite instability has been found mostfrequently in HNPCC, other non-HNPCC tumors contain occasionalalterations. These changes usually involve only one locus, rather thanthe multiple loci typically seen in HNPCC patients (A. Merlo, et al.,supra; R. Wooster, et al., supra). The widespread instability found inSCLC is an exception, and the genetic basis for this is still unknown.In this study, none of the tumors we tested were HNPCC-associated.Comparison of dinucleotide repeat (29 of 4171 tested) versus tri- ortetranucleotide repeat alterations (44 of 874 tested) in these 97 tumorsreveals a significant susceptibility to genetic instability in thelarger alleles (p=0.08×10⁻⁹ by χ² analysis). The high frequency ofmicrosatellite alterations found here suggests that certain loci may beinherently more unstable than others. The mechanism producing thealtered alleles in these tumors may differ from that described in HNPCC(F. Leach, et al., Cell, 75:1215, 1993; R. Fishel, et al., Cell,75:1027, 1993) or could reflect more subtle defects in similar orrelated repair pathways (P. Modrich, Annu. Rev. Genet., 25:229, 1991).From these data, it appears that occasional microsatellite alterationsmay be a relatively generally phenomenon in many human cancers with someloci altered in a tumor-type specific manner.

TABLE 1 FREQUENCY OF MICROSATELLITE ALTERATIONS REPEAT NAME REPEAT HEADAND NECK NSCLC SCLC BLADDER Total SIZE (CHROMO) SEQUENCE SCC (n = 35) (n= 23) (n = 10) TCC (N = 32) (n = 100) Trinucleotide ARA(X) (AGC) 2(6%)1(4%) — 1(3%) 4(4%) D14S50(14) (TCC)7, 1(3%) 1(4%) 0 0 2(2%) (TC)9(AC)6, G (CA)12.5 AR(X)* (CAG)19 6(18%) 1(4%) 4(40%) 1(3%) 2(2%) (CAA)MD(19)* (CTG) 0 1(4%) — 2(6%) 3(3%) SAT(6)* (CAG) 4(11%) 1(4%) — 1(3%)6(6%) DRPLA(12)* (CAG) 1(3%) 0 — 0 1(1%) Tetranucleotide ACTBP2(6)(AAAG)11, 1(3%) 1(4%) 2(20%) 3(9%) 7(7%) (AAAG)15 FGA(4) (TCTT)13 2(6%)1(4%) 0 3(9%) 6(6%) UT762(21) (AAAG) 0 1(4%) 4(40%) 5(15%) 10(10%) Total# of tumors with 10(29%) 2(9%) 5(50%) 9(28%) 26(26%) alterations in{circumflex over ( )}locus Microsatellite alterations for each markertested a number of alterations detected in each tumor type. SCC: headand neck squamous cell carcinoma. NSCLC: non-small cell lung carcinoma.SCLC: small cell lung carcinoma. TCC: transitional cell carcinoma of thebladder. 26 of 100 (26%) total tumors displayed alterations in at leastone locus. *Disease related.

Example 3 Detection of Clonal Populations of Tumor-Derived Cells

These clonal microsatellite alterations were studied to see if theycould be detected in cytologic samples as tumor-specific markers. Todemonstrate this potential clinical application, several correspondingcytological samples that were considered negative for the presence ofcancer cells by light microscopy were analyzed. When DNA from bladdertumor B27 was screened with the tetranucleotide marker FGA, a novel bandwas identified in the tumor lane as compared to normal (FIG. 2A). FIG. 2shows detection of clonal microsatellite alterations in clinicalsamples. PCR conditions and gel analysis have been described (P. van derRiet, et al., supra). Novel bands are indicated by arrows. Panel A), Thecorresponding cytologic urine sample of B27 (TCC) was analyzed usingmarker FGA and compared with normal and tumor DNA. A novel or “shifted”band is seen in the tumor lane vs. the normal lane and at a lesser, butsignificant intensity in the urine lane. Panel (B), Lymphocytic DNA wasnot available from this patient, but a light band is clearly identifiedin the “negative” histological margin of L31 (NSCLC) which correspondsto the more intense novel band in the tumor lane when screened withmarker AR. Panel (C), Amplification of marker CHRNB; the correspondingsputum sample of L25 (SCLC) revealed two light bands consistent with thenovel bands in the tumor lane. These bands are not present in the normalDNA. (N: normal DNA, U: urine DNA, T: tumor DNA, M: margin DNA, S:sputum DNA).

DNA obtained from the patient's urine sample before surgery was screenedwith the same marker, revealing the same novel band at a lower intensityin the urine DNA (D. Sidransky, et al., supra). The intensity of theshifted band was approximately 5% of the intensity of the correspondingband in tumor DNA, indicating that only a small population of cells inthe urine sample was tumor-derived. One patient with SCLC was found tohave a novel CHRNB allele in the tumor DNA when compared to normal DNA.When DNA from a corresponding, prospectively collected sputum sample wasscreened with the marker CHRNB, the identical genetic change in thesputum DNA initially found in the patient's primary SCLC (FIG. 2 c).Again, the lower intensity of the novel bands in sputum suggested thatonly a small fraction of cancer cells was present in the sample. Severalhistopathologically negative surgical margins were examined (D.Sidransky, et al., supra). The tumor DNA of L29 demonstrates a novel,smaller band at the microsatellite marker IFN, while paired DNA of ahistologically negative surgical margin presented the same shifted bandat lower intensity (FIG. 2B). This is consistent with the previousobservation that undetected infiltrating tumor cells can be identifiedin surgical margins after “complete” surgical resection with sensitivemolecular techniques.

These examples demonstrate the ability to detect clonal populations oftumor-derived cells in cytologic samples and histopathologic tissue.This assay was readily reproducible using other altered markers to testcorresponding samples in these cases and paired samples from otherpatients. Clinical samples from patients without a microsatellitealteration detected in the primary tumor were consistently negative.

Example 4 Sensitivity Determination by Dilution

An obvious problem in screening clinical samples is the need to detectan extremely small number of cancer cells among a large background ofnormal cells, especially in bodily fluids such as urine and sputum. Todemonstrate the sensitivity of the method of the invention, the DNA ofbladder tumors B17, which has a larger novel allele, and B27, which hasa smaller novel allele were utilized. These two samples were chosenbecause of our observation that smaller alleles tend to amplify betterthan larger alleles by PCR. Tumor DNA was diluted with normal,lymphocytic DNA from the same patient and 50 ng of DNA from eachdilution was amplified by PCR.

FIG. 3 shows sensitivity determination by simple dilution. DNA fromtumors (T) containing alterations were diluted with the correspondingpatients' lymphocyte DNA (N) from 1 in 5 (20%) to 1 in 1000 (0.1%).Samples were then amplified by PCR with marker FGA, separated bydenaturing gel electrophoresis and visualized by autoradiography asdescribed (P. van der Riet, et al., supra). Novel bands are indicated byan arrow. Panel (A) shows the novel band seen in the tumor lane of B17(TCC) is still visible when diluted with its normal corresponding DNA to0.1%. Similarly in Panel (B), the novel band in the tumor DNA of B27(TCC) is clearly seen when diluted to 0.5%.

The “shifted” band was seen in the dilution mixture containing only 0.1%of tumor DNA in B17 and 0.5% tumor DNA in B27. These results suggestthat this method may potentially detect one cancer cell among 200 to1000 normal cells, thus proving its potential usefulness as a clinicalscreening assay.

Example 5 Multiplex PCR Assay for Detection of Hypermutable Nucleic Acid

Microsatellite alterations in HNPCC and SCLC could be detected by usingjust a few microsatellite repeat markers since these tumors alter a highpercentage of all tested alleles. For non-HNPCC tumors, a singlewell-selected tri- or tetranucleotide repeat marker could potentiallyidentify over 15% of tumors for a particular cancer type. In SCC andTCC, seven selected markers detected over 28% of tumors (TABLE 1).Because the genetic mechanism underlying these occasional alterations isstill not known, it is possible that some cancers will not display anyalterations regardless of how many markers are tested. Nevertheless, thehuman genome contains over 100,000 repeat regions and it is very likelythat other candidate markers could be identified for this analysis. Acombination of repeat markers could be utilized to identify a highpercentage of cancer patients; the ability to potentially test severalmarkers in a single PCR reaction could simplify the ultimate screeningapproach.

DNA from B27 (TCC) and a corresponding urine sample was amplified withthree different primers sets (FGA on chromosome 4, ACTBP2 on chromosome6, and AR on the X chromosome) in the same PCR reaction, separated ondenaturing acrylamide gels and exposed to film. The concentration ofeach primer was diluted to 100 ng/lg in the final PCR reaction. A novelband in the tumor lane is identical to the corresponding, less intenseband in the urine DNA lane. (N: normal DNA, T: tumor DNA, U: urine DNA).

The feasibility of this approach is shown in FIG. 4, where the threemarkers are multiplexed in a single PCR reaction. The results clearlydemonstrate a novel band in urine DNA identical to the altered allele inthe corresponding primary TCC tumor.

This assay is much simpler to perform that previous PCR-based assaysfollowed by cloning and oligomer-specific hybridization to detectoncogene mutations (D. Sidransky, et al., supra). Although thesensitivity of this assay is slightly diminished, with cancer celldetection limited to a background of approximately 500 normal cells ascompared to 10,000 normal cells using the previous approach, evidencefrom prior studies suggests that this is sufficient to detect cancercells in most clinical specimens including sputum (FIG. 3). Moreover,rare oligoclonal events perhaps secondary to inflammation or hyperplasiawould not be detected because of their dilution among an excessbackground of normal cells devoid of these alterations. Althoughoncogene mutations that provide neoplastic cells with a distinct growthadvantage are not specifically detected, monoclonality is a fundamentalcharacteristic of all neoplasms and detection of clonal cell populationsin cytologic samples remains an ominous sign (P. J. Fialkow, Biochem.Biophys. Acta., 458:283, 1976; P. C. Nowell, Science, 94:23, 1976). Theaccumulation of genetic events in subsequent daughter cells is wellrecognized and a detectable clone would be expected to persist andprobably continue along the neoplastic progression pathway (E. R.Fearon, et al., Cell, 61:709, 1990; D. Sidransky, et al., Nature,355:846, 1992; D. Sidransky, et al., N. Engl. J. Med., 326:737, 1992).

Although many of the genetic events in colorectal cancer progression areknown (E. R. Fearon, et al., supra), few events have beenwell-characterized in most other tumor types and not all occur in agiven tumor. The ability to detect early clonal cell populations inpatients without precise knowledge of the specific gene mutations in theprimary tumor is a major strength of this assay. Indeed, these areprecisely the patients that may be ideal candidates for chemopreventivestrategies and/or amenable to surgical resection with careful follow-up.Moreover, detection of rare infiltrating cancer cells in histopathologicmargins may have great impact on current surgical practice. Because theprimary tumor is already resected in these cases, rapid screening oftumor DNA can provide a single marker for detection of theseinfiltrating tumor cells.

The present invention indicates that microsatellite alterations appearto be a common feature in human cancers and that larger repeats areprobably more prone to this type of genetic instability. The highfrequency of alterations observed in several markers appear to betumor-type specific; the selection of appropriate markers with arelatively high rate of instability for a given tumor type may allow theuse of a multiplex PCR test to identify a high percentage of neoplasmsin patients. The identification of these alterations in bodily fluidsand surgical margins attests to their potential use as clonal markers inthe detection of neoplastic cells. A simple and powerful screening testapplicable to a variety of cancers and pathologic samples isdemonstrated by the present invention. Because cancer is so prevalent inthe population, this molecular approach has important implications forcancer detection.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade without departing from the spirit or scope of the invention.

1. A method for detecting a human cell proliferative disorder associatedwith a hypermutable target nucleic acid comprising: isolating thenucleic acid present in a specimen of a human and detecting the presenceof the hypermutable target nucleic acid; wherein the proliferativedisorder is selected from the group consisting of: head and neck smallcell carcinoma (SCC), non small cell lung carcinoma, small cell lungcarcinoma, and bladder transitional cell carcinoma (TCC); and whereinthe hypermutable target nucleic acid is 11 or 15 repeats of thetetranucleotide sequence AAAG in the chromosome 6 microsatellite locusACTBP2; comparing length of the hypermutable target nucleic acid in thespecimen of the human to length of the hypermutable target nucleic acidin a control specimen, wherein a variation in length of the hypermutabletarget nucleic acid between the specimen of the human and the controlspecimen indicates a cell proliferative disorder.
 2. The method of claim1, wherein the hypermutable target nucleic acid is amplified beforedetecting.
 3. The method of claim 2, wherein the amplification is bymeans of oligonucleotides which hybridize to the flanking regions of thehypermutable target nucleic acid.
 4. The method of claim 1, wherein thevariation in length of the hypermutable target nucleic acid is due to anevent selected from the group consisting of a nucleic acid deletion anda nucleic acid addition.
 5. The method of claim 1, wherein the cellproliferative disorder is not due to a repair gene defect.
 6. The methodof claim 1, wherein the specimen of the human is selected from the groupconsisting of sputum, urine, bile, stool, cervical smears, saliva,tears, cerebral spinal fluid, regional lymph node and histopathologicmargins.
 7. The method of claim 3, wherein the nucleotide sequence ofthe flanking region to which the oligonucleotide hybridizes is 5′-TCACTC TTG TCG CCC AGA TT-3′ (SEQ ID NO:17).
 8. The method of claim 7wherein the oligonucleotide is 5′-AAT CTG GGC GAC AAG AGT GA-3′; (SEQ IDNO:37).